1. Field of the Invention
The field of this invention is evaluating human breast duct epithelial cells by cytology to identify breast cancer and breast precancer.
2. Description of the Background Art
Breast cancer is believed to originate in the lining of a single breast milk duct in the breast; and additionally human breasts are believed to contain from 6 to 9 of these ducts. See Sartorius, JAMA 224 (6): 823-827 (1973). For several decades significant members of the medical community dedicated to studying breast cancer have believed and shown that the cytological analysis of cells retrieved from nipple discharge from the breast milk ducts can provide valuable information leading to identifying patients at risk for breast cancer. Indeed, Papanicolaou contributed to the genesis of such a possibility of a xe2x80x9cPapxe2x80x9d smear for breast cancer by analyzing the cells contained in nipple discharge. See Papanicolaou et al, xe2x80x9cExfoliative Cytology of the Human Mammary Gland and Its Value in the Diagnosis of Cancer and Other Diseases of the Breastxe2x80x9d Cancer (1958) March/April 377-409. See also Petrakis, xe2x80x9cPhysiological, biochemical, and cytological aspects of nipple aspirate fluidxe2x80x9d, Breast Cancer Research and Treatment 1986; 8:7-19; Petrakis, xe2x80x9cStudies on the epidemiology and natural history of benign breast disease and breast cancer using nipple aspirate fluidxe2x80x9d Cancer Epidemiology, Biomarkers and Prevention (January/February 1993) 2:3-10; Petrakis, xe2x80x9cNipple Aspirate Fluid in epidemiological studies of breast diseasexe2x80x9d, Epidemiologic Reviews (1993) 15:188-195. More recently, markers have also been detected in nipple fluid. See Sauter et al, xe2x80x9cNipple aspirate fluid: a promising non-invasive method to identify cellular markers of breast cancer riskxe2x80x9d, British Journal of Cancer 76(4):494-501 (1997). The detection of CEA in fluids obtained by a nipple blot is described in Imayama et al. (1996) Cancer 78: 1229-1234.
Despite a long history of nipple aspirate fluid and corresponding cytology, questions have remained about the validity and use of nipple aspirate cytology either for making a diagnosis of breast cancer or breast precancer, or for the correlative value of nipple aspirate fluid cytology to histological and pathological readings from a tissue sample or fine needle aspirate (FNA) cytological readings of the same breast.
Random fine needle aspiration (FNA) of breast tissue has been used to detect lesions in the breast, Fabian et al, J Cell Biochem Suppl 1997; 28-29: 101-10, using cytological analysis of material retrieved from breast by fine needle aspiration. Sample from FNA can be evaluated by a uniform scheme as detailed in Amer. J. Surg, 1997; 174:371-385. FNA as preformed on the breast has several drawbacks. Usually several punctures with an aspiration needle are needed to locate a target lesion; tracks are created with the withdrawal of cancerous cells through the breast with a coordinate risk of spreading the cancer cells to the blood stream along the tracks; scarring can occur at the sites of entry; the punctures can be painful; multiple punctures can result in a sore breast for a time after the procedure; often multiple entries are needed to locate a lesion that provides a positive sample.
Otto Sartorius starting about the mid-1970s obtained some amount of ductal fluid. He accomplished this using a hair thin catheter to infuse saline in the duct, removing the catheter and squeezing the breast or aspirating the nipple surface to get small fluid amounts from the surface. Ductal xe2x80x9cwashingsxe2x80x9d have also been reporter by Susan Love in Love and Barsky, (1996) Lancet 348: 997-999. Susan Love used a catheter to infuse fluid into a breast duct, then removed the catheter after infusing about 1.5 ml of saline, and collected resulting expressed fluid by squeezing the breast and collecting the xe2x80x9cwashingsxe2x80x9d from the nipple surface with a capillary tube.
So described previous attempts at obtaining ductal fluid including nipple aspiration at the nipple surface, either with or without first infusing fluid into the duct, and early attempts at obtaining fluid oozing from the nipple surface after removing the fluid infusion catheter from the duct post fluid infusion have been found inadequate to obtain sufficient cellular yields for a cytological analysis. These samples were not found to be a consistent, uniform, and respected diagnostic tool of breast cancer. Such cytological results, so obtained, have remained ancillary and supportive of other diagnosis (e.g. such as FNA, histology by biopsy, lumpectomy or mastectomy, mammography) and have not been respected as diagnostic information that can stand alone.
The deficiencies of previous nipple aspirate fluid cytology can be mainly attributed to the fact that cellular yields of nipple aspirate fluid are much lower than ductal lavage cell yields when ductal lavage is performed and fluid is retrieved with an indwelling catheter. Petrakis reports that in his studies of some thousands of women over a 20 year study that his average cell yield is about 100 cells per breast, and often less than that using one of the above-mentioned techniques. In a recent clinical trial of about 500 women on whom nipple aspirate fluid was collected prior to ductal lavage with an indwelling catheter, the median number of ductal epithelial cells collected by nipple aspiration was about 120 cells per breast. In addition, samples collected by nipple aspiration tend to yield individual cells, and aggregates or groups of cells of less than 10 cells per aggregate, often less than 5 cells per aggregate, where such groups are collected. Likewise cell yields from these methods using a catheter for fluid infusion of about 1.5 ml of wash fluid, and where the catheter is withdrawn prior to collection of any expressed fluid on the nipple surface, have not yielded cell counts much improved from traditional nipple aspiration.
In contrast, cell yields from ductal lavage samples from infusion of total fluid amounts greater than 2 ml of wash fluid per duct, often greater than 5 ml of wash fluid, commonly as much as in the range from about 10 ml to about 50 ml of wash fluid, and in some cases greater than 50 ml of wash fluid during a lavage procedure on one breast duct where the cells and wash fluid collected are collected while the catheter remains in place in the duct during the procedure have been often greater than 500 cells, commonly from 1000 to 10,000 cells per breast duct, or more per breast duct. In the first trial conducted of its kind with 500 women using a ductal access tool for ductal lavage that permitted infusion of fluid into the breast duct and collection of the fluid mixed with cells while the tool remained in the duct, the mean number of ductal epithelial cells retrieved by ductal lavage was about 40,000 cells/duct and the median number of cells retrieved was about 13,500 cells/duct.
In addition, the cells retrieved by ductal lavage using the methodology of infusion of fluid volumes greater than about 2 ml of saline (e.g. up to about 50 ml or more of fluid during the entire lavage procedure on single duct) and retrieval of the ductal fluid through the lumen of an indwelling catheter, resulted in not only more and larger clumps or clusters of cells, but larger numbers of cells per clump or cluster. Typically, and previously, nipple aspiration yielded clusters or clumps of less than 5 cells per cluster (when clusters are retrieved) and ductal lavage yields clusters or clumps of 10 or more cells per cluster or clump, and substantial numbers of these clumps or clusters. The advantage to cytological readings of clumps or clusters is that they give a cytologist yet another desirable context for relationships between the cells. This context can provide advantages similar to the context derived from a histological analysis where the cells remain in the architecture of the original tissue. The potential for frequent retrieval of cell clumps greater than about 5 cells per clump, especially greater than 10 cells per clump, is a phenomenon not seen in ductal epithelial cell cytology before the ductal lavage techniques so described had been perfected.
In addition, another difference between cells retrieved by ductal lavage and cells retrieved by FNA should be mentioned. Cells retrieved by FNA can include blood from in an around the accessed lesion and can generally be thought of a non-sloughed cells or cells that have not been sloughed from the lesion but rather have been retrieved from a punctured lesion, whereas cells retrieved by ductal lavage (indeed any cells retrieved from the ductal fluid without rupture of the breast tissue, including nipple aspiration, ductal washing collection and ductal lavage) retrieve cells sloughed into the ductal fluid, which cells have left the lesion resident in the breast duct to enter the ductal fluid and be available for retrieval from that fluid source and not from the architecture of the lesion itself as with histology of a biopsy sample or tissue resection and to a lesser extent FNA which retrieves from a lesion but in a more disruptive fashion.
Accordingly, it would be desirable to establish a set of cytological criteria that utilizes, legitimizes and maximizes the new information achievable by newly developed ductal lavage techniques in order to achieve diagnoses of breast cancer and breast precancer that can stand alone and receive acceptance in the medical community without the need for diagnosis from tissue disruption and its coordinate disadvantages.
The invention provides a method of cytologically evaluating epithelial cells collected from a human breast duct, by providing a ductal fluid sample comprising ductal epithelial cells retrieved by ductal lavage of a duct of a breast of a patient, evaluating the ductal epithelial cells in the sample for one or more indicia selected from the group consisting of cell grouping, cell shape, cell size, nuclear size, nuclear shape, presence or absence of nucleoli, nuclear-to-cytoplasmic ratio, vacuoles in the cytoplasm, cytoplasmic shape, cytoplasmic border, presence or absence of anisonucleosis, presence or absence of mitotic figures, nuclear membrane quality, presence of necrotic debris, chromatin distribution, coarseness of chromatin, and the presence or absence of microcalcifications; and classifying the sample as being normal, atypical or malignant based on the observed indicia.
The sample is classified as malignant when the sample is characterized by at least some of an identifying feature selected from the group consisting of a loss of cell cohesiveness, loose clusters of epithelial cells, enlarged cells, enlarged nuclei, high nuclear-to-cytoplasmic ratio, increased cytoplasm in some cells, irregular nuclear membranes, clumped chromatin, hyperchromatic chromatin, unevenly dispersed chromatin, enlarged nucleoli, multiple nucleoli, marked variation among the cells of the sample in cell size and nuclear size, necrotic debris, and microcalcifications in background material appearing as dense material with smooth borders and concentric layers or dystrophic and amorphous.
The sample is classified as atypical with marked changes when the sample is characterized by at least some of an identifying feature selected from the group consisting of enlarged ductal epithelial cells, marked nuclear increase in ductal epithelial cells, variation in size and shape of the ductal epithelial cells as compared to normal ductal epithelial cells, abundant cytoplasm in some cells, decreased nuclear-to-cytoplasmic ratios in some cells, coarse chromatin, mild abnormality in chromatin distribution, larger nucleoli than in normal cells, multiple nucleoli, more prominent nucleoli, nuclei groups that appear to be overlapping, and mitotic figures.
The sample is classified as atypical with mild changes when the sample is characterized by at least some of an identifying feature selected from the group consisting of single ductal cells, cohesive multilayered cells, complex groups of cells, monolayered cells, an increased number of cell layers compared to normal cells, increased overlapping of the cells, nuclear crowding of cells, minimally enlarged cells, moderate increase in nuclear size to within a range from about 12 to about 16 xcexcm in diameter, slight anisonucleosis in some cells, and presence of nucleoli.
The sample is classified as normal when the sample is characterized by at least some of an identifying feature selected from the group consisting of single cells, monolayer sheets, tight cells clusters usually one or two cell layers thick, small nuclei in a size range from about 8 to about 12 xcexcm in diameter, high nuclear-to-cytoplasmic ratio depending on the orientation of the cells in clusters, in single cells a columnar shape of cytoplasm, in single cells discreet small vacuoles in the cytoplasm, in single cells discreet cytoplasmic border, cohesive groups of ductal epithelial cells with cells of uniform size and regular round to oval shape, monolayer sheets of cells with uniform, small cells, monolayer sheets of cells with small inconspicuous nucleoli.
The sample is classified as insufficient cells to make a diagnosis (ICMD) when the sample is characterized by less than 10 epithelial cells in the sample.
The ductal fluid can be retrieved by placing a ductal access tool in the duct and infusing fluid into the duct through the tool and retrieving from the accessed duct through the tool a portion of the infused fluid mixed with ductal fluid that comprises ductal epithelial cells. The method can be repeated for more than one duct on a breast, for a plurality of ducts on a breast. Providing the ductal fluid sample can comprise obtaining the sample from the breast, or receiving a sample which had been previously obtained. The fluid can be obtained by nipple aspiration of the milk ducts.
The fluid sample can be obtained by washing the ductal lumen and retrieving fluid and cells from the lumen. Fluid so obtained can be collected from a single duct, or collected from a plurality of ducts.
The fluid infused can be in a range from about 2 ml to about 100 ml during a total lavage procedure on a single breast duct; the fluid sample retrieved is in a range from about 2ml to about 30 ml of wash fluid mixed with cellular material. The cells retrieved can comprise excess of about 500 cells; the cells retrieved can comprise an amount in a range from about 500 cells to about 40,000 cells from a single breast duct. The sample retrieved can comprise one or more clusters or clumps of cells, wherein a cluster comprises 10 or more ductal epithelial cells.
The method of cytologically evaluating cells collected from a human breast duct can further comprise examining the ductal fluid sample to determine the presence of a marker selected from the group consisting of protein, a polypeptide, a peptide, a nucleic acid, a polynucleotide, an mRNA, a small organic molecule, a lipid, a fat, a glycoprotein, a glycopeptide, a carbohydrate, an oligosaccharide, a chromosomal abnormality, a whole cell having a marker molecule, a particle, a secreted molecule, an intracellular molecule, and a complex of a plurality of molecules.
The invention further provides a system of cytological evaluation of epithelial cells collected from a human breast duct comprising: a tool or apparatus for collecting breast duct fluid from a human breast; a chart or written guidelines for evaluating the ductal epithelial cells in the sample for one or more indicia selected from the group consisting of cell grouping, cell shape, cell size, nuclear size, nuclear shape, presence or absence of nucleoli, nuclear-to-cytoplasmic ratio, vacuoles in the cytoplasm, cytoplasmic shape, cytoplasmic border, presence or absence of anisonucleosis, presence or absence of mitotic figures, nuclear membrane quality, presence of necrotic debris, chromatin distribution, coarseness of chromatin, and the presence or absence of microcalcifications; and an algorithm for classifying the sample as being normal, atypical or malignant based on the observed indicia.
The tool or apparatus can comprise a breast duct access fluid and cell retrieval tool, and one or more of a probe, a tool for administering anesthetic, marking tools for marking an accessed or fluid yielding duct or a collection receptacle for the retrieved fluid and cells.